Photosensitizers are chemicals which kill cells and/or fluoresce when activated by light of a specific wavelength. Most malignant and some premalignant tissues retain these photochemically active substances in higher concentrations and for longer durations than surrounding normal tissues. The retention time is not dependent on wether or not the cells are synthesizing DNA or cell growth or nutrient uptake. This form of treatment, therefore, is an important new part of cancer treatment and tumor detection (Dougherty, T. J., CRC Critical Rev. Oncol. Hematol., 1984, 83).
Photosensitizers have been recognized for almost a century. In 1900, (Rabb, C., Z. Biol., 1900, 39, 1423) reported the lethal effects of a combination of acridin orange dye and ordinary light on Paramecium. In 1903, von Tappeneir reported the first therapeutic use of photosensitizers when he used eosin and white light to treat skin tumors. The phototoxic effect of an administered porphyrin in man was observed in 1913. The localization of administered porphyrins in tumor tissue was recognized in the 1940s. It was not until 1972, however, that these two ideas (photodegradation of tissue and localization in tumors) came together successfully, when Diamond demonstrated that a porphyrin could preferentially degrade tumor implants in a rat (Diamond, I.; McDonagh, A. F.; Wilson, C. B.; Granelli, S. G.; Nielsen, S.; Jaenicke, R., Lancet, 1972, 1175). This result was confirmed and extended by Dougherty, T. J.; Grindey, G. B.; Fiel, R.; Weishaput, K. R.; Boyle, D. G.; J. Natl. Cancer Inst., 1975, 55, 115.
The higher concentration of porphyrins in malignant tumors is used for the treatment and detection of cancer. For detection of early stage small tumors, the porphyrin-containing tumor cells and surrounding tissues are exposed to light. The porphyrins then emit a strong fluorescence, which contrasts with the much weaker fluorescence from the normal tissue, allowing for detection. For the treatment of cancer, photodynamic therapy (PDT) consists of injecting the patient with a photoactive dye and irradiating the tumor area with a wavelength of light which activates the dye to produce toxins which kill the tumor. The porphyrin dyes become toxic to the surrounding environment by producing single oxygen and oxygen radicals (Dougherty, T. J.; Kaufman, J. H.; Goldfarb, A.; Weishaupt, K. R.; Boyle, D.; Mittleman, A.; Cancer Res., 1976, 38, 3628). PDT techniques depend strongly on how well the compound used preferentially concentrates within the tumor cell. Skin photosensitivity is the only known side effect of PDT with certain porphyrin type photosensitizers. Because skin retains these chemicals in enough quantities to produce surface reactions, patients must avoid exposure to sunlight.
The distribution of porphyrin drugs in the body compared with tumor cells is still under investigation. The distribution varies with cell type and porphyrin derivative. It is thought that once the photosensitizer is injected intravenously, some of the drug escapes the blood stream and moves into the interstitial fluid. The porphyrin binds to the cellular membrane and slowly diffuses into the cell cytoplasm. Each porphyrin, then, rapidly binds to hydrophobic regions inside the cell. Fluorescence microscopy of porphyrin-treated leukemia L1210 cells shows a localization around the plasma membrane and within the intracellular vesicles.
Photofrin.RTM., a hematoporphyrin derivative (Dougherty, T. J.; Boyle, D. G.; Weishaupt, K. R., "Photodynamic Therapy--Clinical and Drug Advances, Porphyrin Photosensitization," Plenum Press, New York, 1983, p. 3) is the only photosensitizer currently being used all over the world for the treatment of a variety of solid tumors. Hematoporphyrin derivative (Hpd) is prepared by mixing hematoporphyrin with glacial acetic acid and sulfuric acid, followed by hydrolysis and precipitation under acidic conditions. This method was partially described by Lipson et al (Lipson, R. L.; Baldes, E. J.; Olsen, A. M., J. Natl. Cancer Inst., 1961, 26, 1). Hpd thus produced consists of a variety of porphyrins. When Hpd is separated into its two main fractions by gel filtration with Sephadex LH-20, the higher molecular weight portion, called Photofrin.RTM., is a more efficient PDT agent (Dougherty, T. J.; Boyle, D. G.; Weishaupt, K. R.; Henderson, B.; Potter, W.; Bellnier, D. A.; Wityk, K. E., Adv. Exp. Biol. Med., 1983, 160, 3). The recommended human dosage of Photofrin.RTM. is 1-2 mg/kg of body weight. The main components of Photofrin.RTM. are dimers and higher oligomers linked with ether, and possibly carbon-carbon linkages (Pandey, R. K.; Siegel, M. M.; Tsao, R.; McReynolds, J. M.; Dougherty, T. J., Biomed. and Environ. Mass Spectrometry, 1990, 19, 405).
For a photosensitizer to be clinically useful, it must be non-toxic, selectively taken up and/or retained in malignant tissues, activated by penetrating light (&gt;600 nm), and photochemically efficient. Although Photofrin.RTM. has been approved for commercialization in Canada and is expected to be approved in other countries, including the United States, it lacks rapid clearance from tissues, is a complex mixture of oligomers, and has the disadvantage that its absorbance at 630 nm is not optimized for tissue penetration. New porphyrin photosensitizers are thus needed for the improvement of photodynamic therapy for cancer treatment.
Our search for more efficient, chemically pure, less phototoxic, and better localizing porphyrins was guided by patterns recognized in the variety of new porphyrins which have recently been shown to be successful PDT agents. The important porphyrin and chlorin derivatives which have led to the development of this research have been reviewed by Pandey, R. K.; Majchrzycki, D. F.; Smith, K. M.; Dougherty, T. J., Proc. SPIE, 989, 1065, 104. The aspartyl derivatives of chlorin e.sub.6, monoaspartyl chlorin e.sub.6 and diaspartyl chlorin e.sub.6, were found to be effective photosensitizers in vitro (Roberts, W. G.; Shaiu, F. Y.; Nelson, J. S.; Smith, K. M., Roberts, M. W., J. Natl. Cancer Inst., 1988, 80, 330). With these compounds, the aspartyl group was noted to be responsible for the efficiency of tissue clearance. In pheophorbide, pyropheophorbide and chlorin e.sub.6 series, certain alkyl ether derivatives including 2-(1-hexoloxyethyl)2-des vinyl derivatives were found to be excellent photosensitizers compared with parent compounds, methyl pheophorbide-a, pyropheophorbide- and chlorine.sub.6. (Pandey, R. K.; Bellnier, D. A.; Smith, K. M.; Dougherty, T. J., Photochem. Photobiol., 1991, 53, 65). This was attributed to the increased hydrophobicity of the hexyl group and is consistent with studies done by Evensen on porphyrins with varying polarities (Evenson, J. F.; Sommer, S.; Riminfton, C.; Moan, J., Br. J. Cancer, 1987, 55, 483).
Chang, C. K., Sotiroiu, C.; Wu, W., J. Chem. Soc., Chem. Commun., 1986, 1213, have previously shown that chlorins, on reacting with osmium tetroxide can be converted to vic dihydroxy bacteriochlorin system. We extended this methodology in the pheophorbide-a and chlorin e.sub.6 series, and prepared a series of vic -dihydroxy and keto-bacteriochlorins (Pandey, R. K.; Shiau, F. Y.; Sumlin, A. B.; Dougherty, T. J.; Smith, K. M., Bioorg. & Med. Chem. Lett., 1992, 2, 491). It has also been reported that the regiospecificity of pyrrole subunits in osmium tetroxide oxidation is affected significantly by the presence of electron withdrawing substituents in the macrocycle(5a). These stable bacteriochlorins, prepared from mesochlorin e.sub.6 trimethylester and pyropheophorbide-a methylester, have strong absorption in the red region (730 to 750 nm), but, did not show any significant in vivo photosensitizing activity (Kessel, D.; Smith, K. M.; Pandey, R. K.; Shaiu, F. Y.; Henderson, B., Photochem. Photobiol., 1993, 58, 200).
A few years ago, Hoober, J. K.; Sery, T. W.; Yamamoto, Y., Photochem. Photobiol., 1988, 48, 579 have shown that purpurin-18 2, which has strong absorption at 700 nm might be useful photosensitizer for photodynamic therapy (PDT).
Among long wavelength absorbing photosensitizers, bacteriochlorins have been proposed as potential useful candidates for use in photodynamic therapy (PDT) where strong absorptions in the visible spectrum can be used to photoactivate dyes previously located in targeted (neoplastic) tissues (Pandey, R. K.; Shiau, F. Y.; Isaac, M.; Ramaprasad, S.; Dougherty, T. J.; Smith, K. B., Tetrahedron Lett., 1992, 33, 7815). Some naturally occurring bacteriochlorins, have previously been reported as effective photosensitizers both in vitro and as in vivo (Beems, E. M.; Dubbelman, T. M. A. R.; Lugtenburg, J.; Best, J. A. B.; Smeets, M. F. M. A.; Boehgeim, J. P. J., Photochem. Photobiol., 1987, 639). However, most of the naturally occurring bacteriochlorins (760-780 nm) are extremely sensitive to oxygen, which results in rapid oxidation to the chlorin state (640 nm); thus the spectroscopic properties of the bacteriochlorins are lost. Further, if a laser is used to excite the bacteriochlorin in vivo, oxidation may result in the formation of a new chromophore absorbing outside the laser window, thus reducing the photodynamic efficiency.